In an important type of in situ assay, a cell (or virus) is incubated with a target-specific probe that is either a fluorescent dye or a fluorescent dye attached to a target-specific molecule. Detection is normally accomplished with a flow cytometer or microscope. Such assays are useful to detect viral nucleic acids, human genes of interest, specific cellular antigens, and other biologically important molecules.
In the first steps of a fluorimetric measurement, a cell is exposed to light with a wavelength that the probe dye can absorb. The dye will then emit light at a longer wavelength. A significant problem, however, is that the light emitted by a probe-treated cell will not exclusively originate from fluorescent probe molecules bound to specific target molecules. Rather, some of the light emitted will be from cellular molecules that are not bound to probe molecules. Additionally, some light will be emitted from probe molecules that have bound non-specifically.
Light emitted by cellular molecules (or vital molecules) not bound to probe molecules is referred to as autofluorescence, and such light-emitting molecules are autofluorescing molecules. Autofluorescence occurs at wavelengths in the range 470 nm to 570 nm, as well as above and below that range; a major peak in the spectrum is at about 520 nm. If the emission wavelengths of the probe dye are also in the range 470 nm to 570 nm, then autofluorescence will contribute significantly to the assay background. An example of a commonly used probe dye that emits in that range is fluorescein isothiocyanate ("FITC"). Autofluorescence can, however, also be important when other probe dyes, such as phycoerythrin, are used.
The present invention involves the discovery of compounds useful as background reducers in in situ assays.
In addition to the general benefit of increased assay sensitivity that the background reducers provide, there is the additional advantage that, for a given level of sensitivity to be reached, cell treatment procedures such as cell fixation need not be as detrimental to cell integrity as would be the case without the use of the background reducers. As a result, the cells retain their structural integrity for a longer time, a consideration particularly important for flow cytometry.